Methotrexate is the most commonly used second line agent for the treatment of Rheumatoid Arthritis (RA) and remains the standard to which new drugs are compared. Despite its clear efficacy 30-40 % of patients do not achieve a therapeutic response or develop significant toxicity such as hepatic fibrosis. In the preceding funding period we have demonstrated that methotrexate promotes adenosine release from stressed cells and tissues and that adenosine, acting at its receptors (most notably AzA receptors) on inflammatory cells, suppresses inflammation. In this renewal application we propose two Aims designed to broaden our understanding of the actions of methotrexate. In the current funding period we have demonstrated that Th1 cytokines modulate adenosine receptor expression and signaling and we propose to follow up on these observations and further study the regulation of adenosine receptor expression and function. To this end we will examine Th1 cytokine (TNFalpha, IL1 and IFNgamma)-mediated regulation of expression and phosphorylation of G proteins, expression of protein kinase A, CREB phosphorylation, phosphodiesterase expression and modulation of other downstream regulators of adenosine receptor-stimulated signal transduction. We have previously found evidence using pharmacologic inhibitors that methotrexate promotes, by a mechanism that is only poorly understood, release of adenine nucleotides which are converted to adenosine extracellularly by the action of ecto-5'nucleotidase. To better understand the molecular basis for this phenomenon we will examine methotrexate-mediated regulation of adenosine generation by studying the ability of methotrexate to increase adenosine and adenine nucleotide release and suppress inflammation in animals deficient in nucleoside triphosphate dephosphorylase (NTPDase, CD39) and ecto-5'Nucleotidase (ecto-5'NTase, CD73). Methotrexate is the cornerstone of therapy for RA and other forms of inflammatory arthritis; a better understanding of its mechanism of action and toxicity may permit the design of new, more effective and less toxic agents for the therapy of RA.